Sample buffer

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Sample buffer

Postby newbie17425 » Feb 05 2018 7:57 pm

Hi,

I have 800 µl of sample buffer stocks (without DTT) stored at -20°C. Before using I add 2.5 M DTT and use it.

Sometimes, I add the sample buffer to my samples and store at -20°C. However, the next day after heating the samples, the color of the buffer changes from blue to dark green. But, when I load it to the gels, they appear blue. Why does the color change after heating.

After adding the buffer to the sample, should I store the buffer at -20°C for next day use or should I store at RT. How long can the buffer be stored (days, weeks?) at RT or -20°C?

Thanks.
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Re: Sample buffer

Postby relaxin » Feb 06 2018 12:38 pm

I do not recall any change of color of samples after heating. It could be due to change in pH. I guess it will not affect the sample.

When you add sample buffer to your samples, you need to boil them immediately. Some proteases are active in the presence of SDS. After boiling, the samples should be safe for storage at 4 C overnight. For longer term storage, they should be stored at -20 C. You need to reheat them again prior to loading on gel. This is to make sure everything is re-dissolved.
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Re: Sample buffer

Postby newbie17425 » Feb 06 2018 12:40 pm

relaxin wrote:I do not recall any change of color of samples after heating. It could be due to change in pH. I guess it will not affect the sample.

When you add sample buffer to your samples, you need to boil them immediately. Some proteases are active in the presence of SDS. After boiling, the samples should be safe for storage at 4 C overnight. For longer term storage, they should be stored at -20 C. You need to reheat them again prior to loading on gel. This is to make sure everything is re-dissolved.


So, you suggest heating the sample twice.
If stored at 4°C overnight, do I need to re-heat it?
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Re: Sample buffer

Postby relaxin » Feb 06 2018 12:59 pm

The second heating can be done at 70 C for 2 min. For samples stored overnight at 4 C, SDS may form precipitate, heat at 70 C will re-dissolve it.
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Re: Sample buffer

Postby newbie17425 » Feb 06 2018 1:47 pm

relaxin wrote:The second heating can be done at 70 C for 2 min. For samples stored overnight at 4 C, SDS may form precipitate, heat at 70 C will re-dissolve it.


I am using KCC2 antibody and it has been advised not to heat more than 37 degrees. So do you suggest heatijg it again at 37 degrees for 30 min after incubation at 4 degrees?
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Re: Sample buffer

Postby relaxin » Feb 07 2018 12:10 pm

I think the advice of not to heat the antibody over 37 C is to preserve its function. When you analyze your IP on SDS gel, boiling the sample is to destroy the antibody and release the antigen. I do not think you need to use low temperature.

Of course, the second heating can be done at 37 C, since this is to re-dissolve SDS.
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